All eukaryotic DNA is packaged into a nucleoprotein structure called chromatin. In vitro studies have suggested that the chromatin-associated High Mobility Group (HMG) proteins from higher eukaryotes play a role in nuclear processes such as transcription and/or DNA replication. A genetic approach is necessary to elucidate directly the functions of HMG proteins in the cell. Therefore, we have cloned the genes for two HMG proteins, NHP6A and NHP6B, from Saccharomyces cerevisiae. Specific antisera were also prepared against both proteins. Analysis of deletion mutants in NHP6A and NHP6B provided evidence that these HMG proteins have important, but nonessential, functions in the eukaryotic cell. We are now poised to combine genetic and biochemical approaches to reveal further the roles of these proteins in the cell. We propose to decipher the functions of the NHP6 proteins by biochemically characterizing the cause of the lethal phenotype of cells overexpressing NHP6. Conditional expression of NHP6B has been achieved by placing the gene under the control of an inducible promoter. These cells will be analyzed, after shifting them from permissive (noninducing) to nonpermissive (inducing) growth conditions, for changes in RNA, DNA and protein synthesis as well as for changes in chromatin structure. The results of these experiments will help define the in vivo functions of NHP6. An important direction of this research is to combine the genetic approaches with in vitro studies to elucidate the molecular mechanisms of NHP6 function. As one step, extracts from nhp6a- nhp6b- mutants will be tested in in vitro transcription reactions to assess the possible roles of the NHP6 proteins. In addition, the DNA-binding specificity, if any, of NHP6A and NHP6B will be investigated by nitrocellulose binding and gel mobility shift assays. Lastly, to determine the interactions of the NHP6 proteins with other chromatin proteins two approaches will be used: (1) NHP6-containing complexes will be isolated by immunoprecipitation and the components identified by gel electrophoresis, and (2) genes for proteins interacting with NHP6A/6B will be cloned using a novel genetic system in which beta-galactosidase synthesis depends upon protein-protein interactions. Finally, since the NHP6 proteins are prototypes for a novel DNA- binding motif called the HMG Box, a structure/function analysis of NHP6A will be undertaken. The phenotypes of cells mutated in the condons for the most highly conserved amino acids will be assessed in the assays described above. These experiments will indicate which amino acids are critical for NHP6 function.